This invention is generally in the field of methods of the treatment and prevention of inflammatory responses using peptides derived from selectins including GMP-140, ELAM-1, and lymphocyte-homing receptor.
The adherence of platelets and leukocytes to vascular surfaces is a critical component of the inflammatory response, and is part of a complex series of reactions involving the simultaneous and interrelated activation of the complement, coagulation, and immune systems.
The complement proteins collectively play a leading role in the immune system, both in the identification and in the removal of foreign substances and immune complexes, as reviewed by Muller-Eberhard, H. J., Ann. Rev. Biochem. 57:321-347 (1988). Central to the complement system are the C3 and C4 proteins, which when activated covalently attach to nearby targets, marking them for clearance. In order to help control this process, a remarkable family of soluble and membrane-bound regulatory proteins has evolved, each of which interacts with activated C3 and/or C4 derivatives. The coagulation and inflammatory pathways are regulated in a coordinate fashion in response to tissue damage. For example, in addition to becoming adhesive for leukocytes, activated endothelial cells express tissue factor on the cell surface and decrease their surface expression of thrombomodulin, leading to a net facilitation of coagulation reactions on the cell surface. In some cases, a single receptor can be involved in both inflammatory and coagulation processes.
Leukocyte adherence to vascular endothelium is a key initial step in migration of leukocytes to tissues in response to microbial invasion. Although a class of inducible leukocyte receptors, the CD11-CD18 molecules, are thought to have some role in adherence to endothelium, mechanisms of equal or even greater importance for leukocyte adherence appear to be due to inducible changes in the endothelium itself.
Activated platelets have also been shown to interact with both neutrophils and monocytes in vitro. The interaction of platelets with monocytes may be mediated in part by the binding of thrombospondin to platelets and monocytes, although other mechanisms have not been excluded. The mechanisms for the binding of neutrophils to activated platelets are not well understood, except that it is known that divalent cations are required. In response to vascular injury, platelets are known to adhere to subendothelial surfaces, become activated, and support coagulation. Platelets and other cells may also play an important role in the recruitment of leukocytes into the wound in order to contain microbial invasion.
Endothelium exposed to xe2x80x9crapidxe2x80x9d activators such as thrombin and histamine becomes adhesive for neutrophils within two to ten minutes, while endothelium exposed to cytokines such as tumor necrosis factor and interleukin-1 becomes adhesive after one to six hours. The rapid endothelial-dependent leukocyte adhesion has been associated with expression of the lipid mediator platelet activating factor (PAF) on the cell surface, and presumably, the appearance of other endothelial surface receptors. The slower cytokine-inducible endothelial adhesion for leukocytes is mediated, at least in part, by an endothelial cell receptor, ELAM-1, that is synthesized by endothelial cells after exposure to cytokines and then transported to the cell surface, where it binds neutrophils. The isolation, characterization and cloning of ELAM-1 is reviewed by Bevilacqua, et al., in Science 243, 1160-1165 (1989). A peripheral lymph node homing receptor, also called xe2x80x9cthe murine Mel 14 antigenxe2x80x9d, xe2x80x9cLeu 8xe2x80x9d, the xe2x80x9cLeu 8 antigenxe2x80x9d and xe2x80x9cLAM-1xe2x80x9d, is another structure on neutrophils, monocytes, and lymphocytes that binds lymphocytes to high endothelial venules in peripheral lymph nodes. The characterization and cloning of this protein is reviewed by Lasky, et al., Cell 56, 1045-1055 (1989) (mouse) and Tedder, et al., J. Exp. Med.170, 123-133 (1989).
GMP-140 (granule membrane protein 140), also known as PADGEM, is a cysteine-rich and heavily glycosylated integral membrane glycoprotein with an apparent molecular weight of 140,000 as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). GMP-140 was first purified from human platelets by McEver and Martin, J. Biol. Chem. 259:9799-9804 (1984). The protein is present in alpha granules of resting platelets but is rapidly redistributed to the plasma membrane following platelet activation, as reported by Stenberg, et al., (1985). The presence of GMP-140 in endothelial cells and its biosynthesis by these cells was reported by McEver, et al., Blood 70(5) suppl. 1:355a, Abstract No. 1274 (1987). In endothelial ells, GMP-140 is found in storage granules known as the Weibel-Palade bodies. (McEver, et al. J. Clin. Invest. 84:92-99 (1989) and Hattori, et al., J. Biol. Chem. 264:7768-7771 (1989)). GMP-140 (called PADGEM) has also been reported to mediate the interaction of activated platelets with neutrophils and monocytes by Larsen, et al., in Cell 59, 305-312 (October 1989) and Hamburger and McEver, Blood 75:550-554 (1990).
The cDNA-derived amino acid sequence, reported by Johnston, et al., in Cell 56, 1033-1044 (Mar. 24 1989), and in U.S. Ser. No. 07/320,408 filed Mar. 8, 1989 now U.S. Pat. No. 8,378,464, indicates that it contains a number of modular domains that are likely to fold independently. Beginning at the N-terminus, these include a xe2x80x9clectinxe2x80x9d domain, an xe2x80x9cEGFxe2x80x9d domain, nine tandem consensus repeats similar to those in complement binding proteins, a transmembrane domain (except in a soluble form that appears to result from differential splicing), and a cytoplasmic tail.
When platelets or endothelial cells are activated by mediators such as thrombin, the membranes of the storage granules fuse with the plasma membrane, the soluble contents of the granules are released to the external environment, and membrane bound GMP-140 is presented within seconds on the cell surface. The rapid redistribution of GMP-140 to the surface of platelets and endothelial cells as a result of activation suggested that this glycoprotein could play an important role at sites of inflammation or vascular disruption.
This important role has been confirmed by the observation that GMP-140 is a receptor for neutrophils (Geng et al., Nature 343:757-760 (1990); Hamburger and McEver, Blood 75:550-554 (1990)), monocytes (Larsen, et al., Cell 59:305-312 (1989); Moore, et al., J. Cell Biol. 112:491-499 (1991)), and perhaps a subset of lymphocytes (Moore, et al., J. Cell Biol. 112:491-499 (1991)). Thus, GMP-140 can serve as a receptor for leukocytes following its rapid mobilization to the surfaces of platelets and endothelial cells stimulated with agonists such as thrombin. This role in leukocyte recruitment may be important in hemostatic and inflammatory processes in both physiologic and pathologic states.
Peptides derived from GMP-140 are described in U.S. Ser. No. 07/554,199 entitled xe2x80x9cFunctionally Active Selectin-Derived Peptidesxe2x80x9d filed Jul. 17, 1990 by Rodger P. McEver now abandoned, that are useful in diagnostics and in modulating the hemostatic and inflammatory responses in a patient wherein a therapeutically effective amount of a peptide capable of blocking leukocyte recognition of GMP-140 is administered to the patient. U.S. Ser. No. 07/554,199 filed Jul. 17, 1990 now abandoned, also discloses that peptide sequences within the lectin domain of GMP-140, having homology with the lectin domains of other proteins, especially ELAM-1 and the homing receptor, selectively inhibit neutrophil adhesion to purified GMP-140, and can therefore be used in diagnostic assays of patients and diseases characterized by altered binding by these molecules, in screening assays for compounds altering this binding, and in clinical applications to inhibit or modulate interactions of leukocytes with platelets or endothelial cells involving coagulation and/or inflammatory processes.
ELAM-1, the homing receptor, and GMP-140 have been termed xe2x80x9cselectinsxe2x80x9d, based on their related structure and function. ELAM-1 is not present in unstimulated endothelieum. However, when endothelium is exposed to cytokines such as tumor necrosis factor or interleukin-1, the gene for ELAM-1 is transcribed, producing RNA which in turn is translated into protein. The result is that ELAM-1 is expressed on the surface of endothelial cells one to four hours after exposure to cytokines, as reported by Bevilacqua et al., Proc.Natl.Acad.Sci.USA 84:9238-9242 (1987) (in contrast to GMP-140, which is stored in granules and presented on the cell surface within seconds after activation). ELAM-1 has been shown to mediate the adherence of neutrophils to cytokine-treated endothelial and thus appears to be important in allowing leukocytes to migrate across cytokine-stimulated endothelium into tissues. The cDNA-derived primary structure of ELAM-1 indicates that it contains a xe2x80x9clectinxe2x80x9d domain, an EGF domain, and six (instead of the nine in GMP-140) repeats similar to those of complement-regulatory proteins, a transmembrane domain, and a short cytoplasmic tail. There is extensive sequence homology between GMP-140 and ELAM-1 throughout both proteins, but the similarity is particularly striking in the lectin and EGF domains.
Homing receptors are lymphocyte surface structures that allow lymphocytes to bind to specialized endothelial cells in lymphatic tissues, termed high endothelial cells or high endothelial venules (reviewed by Yednock and Rose, Advances in Immunology, vol. 44, F. I. Dixon,ed., 313-378 (Academic Press, New York 1989). This binding allows lymphocytes to migrate across the endothelium into the lymphatic tissues where they are exposed to processed antigens. The lymphocytes then re-enter the blood through the lymphatic system. The homing receptor contains a lectin domain, an EGF domain, two complement-binding repeats, a transmembrane domain, and a short cytoplasmic tail. The homing receptor also shares extensive sequence homology with GMP-140, particularly in the lectin and EGF domains.
Based on a comparison of the lectin domains between GMP-140, ELAM-1, and the homing receptor (LEU-8), it may be possible to select those peptides inhibiting binding of neutrophils to GMP-140 which will inhibit binding of ELAM-1, the homing receptor, and other homologous selecting, to components of the inflammatory process, or, conversely, which will inhibit only GMP-140 binding.
The in vivo significance of platelet-leukocyte interactions has not been studied carefully. However, in response to vascular injury, platelets are known to adhere to subendothelial surfaces, become activated, and support coagulation. Platelets and other cells may also play an important role in the recruitment of leukocytes into the wound in order to contain microbial invasion. Conversely, leukocytes may recruit platelets into tissues at sites of inflammation, as reported by Issekutz, et al., Lab. Invest. 49:716 (1983).
The coagulation and inflammatory pathways are regulated in a coordinate fashion in response to tissue damage. For example, in addition to becoming adhesive for leukocytes, activated endothelial cells express tissue factor on the cell surface and decrease their surface expression of thrombomodulin, leading to a net facilitation of coagulation reactions on the cell surface. In some cases, a single receptor can be involved in both inflammatory and coagulation processes.
Proteins involved in the hemostatic and inflammatory pathways are of interest for diagnostic purposes and treatment of human disorders. However, there are many problems using proteins therapeutically. Proteins are usually expensive to produce in quantities sufficient for administration to a patient. Moreover, there can be a reaction against the protein after it has been administered more than once to the patient. It is therefore desirable to develop peptides having the same, or better, activity as the protein, which are inexpensive to synthesize, reproducible and relatively innocuous.
It is preferable to develop peptides which can be prepared synthetically, having activity at least equal to, or greater than, the peptides derived from the protein itself.
It is therefore an object of the present invention to provide peptides interacting with cells recognized by selectins, including GMP-140, ELAM-1, and lymphocyte homing receptor.
It is another object of the present invention to provide methods for using these peptides to inhibit leukocyte adhesion to endothelium or to platelets.
It is a further object of the present invention to provide methods for using these peptides to modulate the immune response and the hemostatic pathway.
It is yet another object of the present invention to provide peptides for use in diagnostic assays relating to GMP-140, ELAM-1, and lymphocyte homing receptor.
Peptides derived from three regions of the lectin domain of GMP-140 and the related selecting, ELAM-1 and the lymphocyte homing receptor, have been found to inhibit neutrophil adhesion to GMP-140. These and additional peptides have been synthesized having the following formula:
R1-X-A-B-C-D-E-Y-R2xe2x80x83xe2x80x83(I)
or a pharmaceutically acceptable acid- or based-addition salt thereof wherein:
A is D- or L-asparagine, D- or L-isoleucine or D- or L-valine;
B is D- or L-asparagine or glycine;
C is D- or L-lysine, D- or L-valine or glycine;
D is D- or L-valine, D- or L-threonine or D- or L-isoleucine;
E is D- or L-tryptophan;
X and Y are linear chains of from 0 to 10 amino acids;
R1 is H (signifying a free N-terminal group), formyl, lower alkyl, aryl, lower alkanoyl, aryl, alkyloxycarbonyl or aryloxycarbonyl and
R2 is OH (signifying a free C-terminal group) lower alkyl or aryl esters, or NR3R4 where R3 and R4 each selected independently from H, lower alkyl or aryl.
Peptides of Formula I have as their core region portions of the 56-60 amino acid sequence of GMP-140, with residue 1 defined as the N-terminus of the mature protein after the cleavage of the signal peptide. Examples of peptides of Formula I demonstrate the inhibition of the binding of neutrophils to GMP-140 in concentrations ranging from 5 to 1500 xcexcM. It has been found that alterations within the core sequence, as well as N-terminal and C-terminal flanking regions, do not result in loss of biological activity.
The peptides are useful as diagnostics and, in combination with a suitable pharmaceutical carrier, for clinical applications in the modulation or inhibition of coagulation processes or inflammatory processes.